As a result of confirming the optimal concentration to cause acanthamoebic keratitis (AK) in mice, It was observed that the results of infection with 0.3125 × 105 and 0.1 × 105 of A. castellanii were not constant in repeated experiments. The sufficient number of amoebas that can cause mouse eye keratitis was 0.5 × 105 amoebas. Finally, 0.5 × 105 amoebas on 2 mm lens were inoculated into mice eyes, and the keratitis symptoms were observed from day 1 to 7 post-inoculation. white ring corneal infection, circular edema the mouse eye was observed on day 1, which progressed to corneal infection on day 2 to 7. On the other hand, the sham-operated mice did not show the keratitis symptoms (Fig. 11A).
To confirm the cause of AK development in mice, PCR products from DNA samples of mouse eyeballs on day 1 to 7 post-inoculation were obtained, giving an amplicon of 1,080 bp size, corresponding to 18S-rRNA gene, which was same as that of A. castellanii used as a positive control. On the other hand, the same PCR product from normal and sham-operated mouse DNA was not amplified (Fig. 11B).
PCR products amplified with P-FLA primers were subjected to sequence analysis and gene homology search. On comparing with the 18S-rRNA of A. castellanii, the amplified bands on day 1, 2, 3, 5 and 7 showed 96, 97, 97, 91, 94 and 93 % homology, respectively (Fig. 12).
32
33
Fig. 11. Observation of acanthamoebic keratitis infection with 0.5 × 105 A.
castellanii, and 18S-rRNA amplification on mice eye. (A) Total 0.5 × 105 A.
castellanii (trophozoites with cysts mixed equally) applied on the contact lens were inoculated into mice eye; AK symptoms were observed from day 1 to 7 (S: sham operation, E: experiment). (B) To confirm the causative agent of AK, genomic DNA extracted from the eye tissues on day 1, 2, 3, 4, 5, 6, and 7 was subjected to PCR with P-FLA primers for the amplification of 18S-rRNA gene. A. cast, genomic DNA of A.
castellanii trophozoites (1 × 106) used as a positive control; Normal, DNA extracted from normal mouse eye tissue as a negative control; Sham, DNA extracted from the sham-operated eye tissues as a negative control. Exp I, II, III, experiments were practice three times. M, size marker (below bands figure).
34
35
36
Fig. 12. The nucleotide sequence and homology analysis of 18S-rRNA gene obtained from keratitis-induced mouse eye. PCR products (bands figures on left) on day 1 (A), 2 (B), 3 (C), 4 (D), 5 (E), 6 (F), and 7 (G) post-inoculation were sequenced and confirmed by NCBI BLAST search (right box), and the homology was analyzed with BLAST search (below box).
37
F. Effect of commercial contact lens solutions on the AK development in mice
(1) Morphology of A. castellanii pre-treatment with solution A or B
To investigate the effect of the commercial lens solutions, 0.5 × 105 A.
castellanii were treated with either solution A or solution B, and cultured for 1, 6, 12, or 24 h. On the observation with an inverted microscope, round morphological shapes were observed from 1 to 6 h, some pre-cystic form appeared at 12 h, and cysts were observed at 24 h (Fig. 13A, B).
38
39
Fig. 13. Morphological change of A. castellanii (trophozoites and cysts) treated with commercial lens solution A or B. A. castellanii (trophozoite and cyst, mixed equally) were cultured in commercial lens solution A (A), or B (B) for 1, 6, 12 and 24 h. Arrows indicate pre-cyst and cyst. Scale bar, 20 μm.
40
(2) Change of acanthamoebic keratitis development in mice
To investigate the effect of the commercial contact lens solution, 0.5 × 105 of A.
castellanii (trophozoites mixed equally with cysts) pre-treated with solution A or B were inoculated into mouse eye, and keratitis symptoms were observed for 1, 2, 3, 5, or 7 days (Fig. 14).
As a result of the pre-treatment for 1 or 6 h, solution A and B did not show any change in mice eyeball, in comparison with the established AK mouse model (Result E in this paper). Very weak keratitis symptoms were observed on day 1, and a progressive corneal infection on day 2 to 5. On the 7th day, it was confirmed that the typical ulcer circle had worsened (Fig. 14, 15). Subsequently, it was confirmed by PCR with P-FLA primers with an amplicon of the same size (1080 bp) as that of A. castellanii. In addition, all PCR products were sequenced and identified as A. castellanii 18S-rDNA gene (data not shown). On the other hand, control mice (0.5 × 105 amoeba infected) showed the keratitis symptoms.
On pre-treatment of lens with solution A or B for 12 h, the mice did not show the keratitis symptoms on day 1, even though a weak corneal infection was present (Fig. 14, 15). A mild infection was observed on day 2 and 3, and the typical ulcer circle developed on day 5 and 7 (Fig. 14, 15). The results were also confirmed by PCR with P-FLA primers (Fig. 14, 15) and DNA sequencing (data not shown). On the other hand, control mice (0.5 × 105 amoeba infected) showed the keratitis symptoms (Fig. 14, 12h).
As a result of the pre-treatment of lenses with solution A or B for 24 h, keratitis
41
symptoms were not observed on day 1 and 2. A mild infection was observed on day 3 and the circular edema with increased central corneal involvement developed on the day 5 and 7 (Fig. 14, 15), which was same as the previous established AK mouse model (Result E) (Fig. 14, 15). Subsequently, it was also confirmed by PCR with P-FLA primers (Fig. 14, 15) and DNA sequencing (data not shown). Otherwise, keratitis symptoms observed in control mice (0.5 × 105 amoeba infected) (Fig. 14, 15).
42
43
Fig. 14. Observation of acanthamoebic keratitis upon pre-treatment with solution A. Solution A was used to treat 0.5 × 105 A. castellanii (trophozoite and cyst equally mixed) for 1, 6, 12 and 24 h. AK was induced in mouse eyes and observed for 1, 2, 3, 5 and 7 days (Sham: sham operation). Genomic DNA of mouse eye tissues on day 1, 2, 3, 4, 5 and 7 was subjected to PCR with P-FLA primers for the amplification of 18S-rRNA gene. A. cast, genomic DNA of A. castellanii trophozoites (1 × 106) as a positive control;
Normal, DNA extracted from normal mouse eye tissue as a negative control; (+) control, DNA extracted from AK-mouse eye tissue (inoculated 0.5 × 105 amoeba) as a positive control. M, size marker (below band figures).
44
45
Fig. 15. Observation of acanthamoebic keratitis upon pre-treatment with solution B. Solution B was used to treat 0.5 × 105 A. castellanii (trophozoite and cyst equally mixed) for 1, 6, 12 and 24 h. AK was induced in mouse eyes and observed for 1, 2, 3, 5 and 7 days (Sham: sham operation). Genomic DNA of mouse eye tissues on day 1, 2, 3, 5 and 7 was subjected to PCR with P-FLA primers for the amplification of 18S-rRNA gene. A. cast, genomic DNA of A. castellanii trophozoites (1 × 106) as a positive control;
Normal, DNA extracted from normal mouse eye tissue as a negative control; (+) control, DNA extracted from AK-mouse eye tissue (inoculated 0.5 × 105 amoeba) as a positive control. M, size marker (below band figures).
46
IV. DISCUSSION
Ubiquitous free-living A. castellanii and A. polyphaga cause acanthamoebic keratitis (AK) in animals and in humans, and 85–88% of patients with AK use contact lenses. Furthermore, the number of AK patients is increasing because of the increasing use of contact lenses worldwide (Cerva et al., 1973; Lyons and Kapur, 1977; Brown et al., 1982; Auran et al., 1987; Visvesvara and Stehr-Green, 1990; De Jonckheere, 1991;
Paszko-Kolva et al., 1991; Abjani et al., 2016).
Contact lens users are mainly exposed to AK because Acanthamoeba species can attach themselves to the soft surface of contact lenses and the cornea, resulting in progression of infection. The amoebae attached to the surface of the cornea destroy epithelial cells, penetrate the corneal stroma, and cause infection deep inside the stroma (Hadas and Mazur, 1993; Illingworth et al., 1995; Cao et al., 1998; Cho et al., 2000;
Marciano-Cabral and Cabral, 2003; Por et al., 2009). The symptoms of AK include photophobia, ring-like stromal infiltrates, ulceration, development of retinal lesions, epithelial defects, lid edema, and severe pain (Kilvington and Larkin, 1990; Lorenzo-Morales et al., 2015).
Since AK is mostly chronic, early diagnosis and treatment are important.
Sometimes, AK is misdiagnosed as a fungal or herpes simplex infection, which only involves 10–23% of coinfection by Acanthamoeba (Bacon et al., 1993; Tu et al., 2008b;
Szentmary et al., 2012; Bouheraoua et al., 2013). Currently, there is no effective
47
treatment for AK; imidazole, voriconazole, polymyxin B, and polyhexamethylene biguanide as well as mixed drugs that contain antibiotics, antifungals, antiprotozoals, and antivirals are mainly prescribed (Lim et al., 2008; Dart et al., 2009; Lorenzo-Morales et al., 2013).
Furthermore, preventing AK infection is important, which can be done by keeping the contact lens case clean and not wearing lenses while swimming or showering. In addition, exposure to contaminated water and injury to the cornea should be avoided (Maycock and Jayaswal, 2016).
Although there has been some progress in research regarding the infection mechanism of AK in vitro, in vivo studies using animal models have rarely been conducted. In addition, in these previous studies, AK was induced by direct intrastromal injection of the amoebae into mouse cornea using a microneedle (Matthaei et al., 2012).
However, this procedure is complicated because the intrastromal cornea needs to be injected precisely, for which ophthalmologists are required. Moreover, this method of inducing keratitis is artificial. Therefore, in this study, I attempted to construct an animal model of AK which can be used easily and quickly. In this model, AK can be induced in mice using contact lenses in a manner similar to that of natural infection.
Other previous studies used pigs, rabbits, and rats as animal models for AK (Alizadeh et al., 1995; Said et al., 2004; Awwad et al., 2007; Vural et al., 2007). In this study too, experiments were first performed using rats. However, these animals were difficult to handle, only a limited number of the animals were available, and the cornea
48
was too thick for AK induction (data not shown). Therefore, the experiment was conducted with mice, which are widely used, cheap, and easy to handle and can be used in large numbers in experiments (Ren and Wu, 2010).
In this study, I constructed an AK mouse model by mixing trophozoite and cyst forms of A. castellanii because in natural AK infections, both trophozoite and cyst forms of A. castellanii are detected in contact lenses. The eyes of the mice were scratched with a syringe needle and ophthalmic blade during AK induction because it is difficult to infect the cornea without any damage. Previous studies have shown that AK incidence is affected by corneal damage before infection (van Klink et al., 1993).
After scratching the mouse eyeball, contact lenses with A. castellanii were placed on the eyes. Next, mouse eyelids were sutured. This is because it is difficult to naturally attach contact lenses to mouse eyeballs as they are rounder than human eyeballs, and it was necessary to ensure complete attachment of the lenses to the cornea.
To detect the incidence of AK symptoms, the eyelid sutures were sequentially removed on the days of observation.
To determine the minimum number of Acanthamoeba necessary to induce AK in mice, equal numbers of trophozoites and cysts of A. castellanii were mixed and serially diluted from 1 × 106 to 0.1 × 105 to induce AK. Infection was induced with a minimum of 0.3 × 105 and 0.1 × 105 cells. However, it was not the same as the result of grossly observation and PCR results using the ocular DNAs that caused keratitis in repeated experiments. Therefore, 0.5 × 105 was determined to be the most stable number
49
of A. castellanii for AK induction. After AK induction, symptoms were observed for up to two months and were confirmed to persist and worsen (data not shown). AK induction experiments were at least practiced over three times. Therefore, it was determined that 0.5 × 105 A. castellanii can be used to establish an AK mouse model.
The diagnosis of AK is mainly based on in vitro experiments, in which lenses and lens solution in lens case of patients with AK are cultured or corneal biopsies are performed (Stothard et al., 1998; Stothard et al., 1999; Khan, 2001; Schuster and Visvesvara, 2004a; Qvarnstrom et al., 2006). For accurate diagnosis, PCR can also be used to confirm the amplification of Acanthamoeba DNA (Stothard et al., 1998;
Stothard et al., 1999; Khan, 2001; Schuster and Visvesvara, 2004a; Qvarnstrom et al., 2006; Tu et al., 2008a; Tu et al., 2008b). In this study, eye tissues and contact lenses of the mice induced with AK were cultured on PYG medium and non-nutrient (NN) plates.
However, the presence of A. castellanii was not confirmed because of rapid fungal growth, even after treatment with antifungal reagents. In addition, the number of amoebae remaining in the tissues and contact lenses was too small to culture. Next, eyes of mice that developed keratitis were stained with hematoxylin-eosin (HE) to confirm the presence of amoebae between the cornea and underneath the cornea. However, A.
castellanii was not observed on the mouse eye balls (Fig. 17). It is impossible to observe amoebae under these conditions because the cornea of AK mice is too thin, and it would be difficult to obtain a tissue specimen.
50
51
Fig. 16. Histology of AK-induced mouse eye.
Corneal stroma and polymorphic inflammatory infiltrates consisting of many lymphocytes were observed on tissue slides with Hematoxylin-eosin staining (100 µm and 200 µm)
52
To address these problems, DNA was extracted from the eye tissue of the AK mice, and PCR was performed with P-FLA primers that amplify the 18S rRNA of Acanthamoeba (Tsvetkova et al., 2004). PCR of DNA from the AK mouse tissue produced an amplicon of the same size (1080 bp) as that of Acanthamoeba 18S-rRNA, and sequence analysis of the obtained PCR products revealed that AK was indeed induced by A. castellanii. The bands below 750 bp were identified as the 18S rRNA of Mus musculus.
Contact lens users mostly use commercial multiple lens solutions. However, commercially available solutions are ineffective in preventing infections because of their poor amoebicidal effects (Radford et al., 2002; Kilvington et al., 2004; Joslin et al., 2006; Lorenzo-Morales et al., 2013). It is known that the effects of antibiotics, antifungal agents, and antiviral agents in commercial lens solutions may affect the trophozoites of the Acanthamoeba but have little effect on the cyst form (Zanetti et al., 1995; Hurt et al., 2001; Hiti et al., 2002). Therefore, to confirm the effectiveness of the AK mouse model established in the present study, the effects of commercial lens solutions A and B, which are widely used worldwide, on AK incidence were tested.
After pretreatment with 0.5 × 105 A. castellanii (equal number of trophozoites and cysts) with commercial lens solutions A or B for 1, 6, 12, and 24 h, the shape of all pretreated amoebae changed to that of the pre-cyst and cyst types. The number of amoebae was counted over time to determine if the commercial lens solutions affected amoeba population, but no significant differences were observed between 1 to 24 h (data not
53
shown). Then, the AK mouse model was used to test whether commercial lens solutions A or B could inhibit AK over time. AK mice treated with solutions A or B showed delayed development of AK between day 1 and day 3, but AK was induced in mice treated with solutions A or B by day 5 and day 7. These results indicated that the use of commercial lens solutions may be effective in the short term, but they cannot inhibit AK induction over a long period of use.
In this study, an AK mouse model was established. This mouse model will be an important platform for in vivo testing of AK, for in vitro molecular biology, pathology, and immunology studies, and for the development of new pharmacological drug agents for the treatment of AK.
54
V. CONCLUSION
Acanthamoeba castellanii causes acanthamoebic keratitis (AK). To decipher the exact pathogenesis of AK and development of therapeutic drugs, in vivo model needs to be established. In this study, BALB/c mouse cornea was scratched using a syringe needle and ophthalmic surgical blade under anesthesia. A. castellanii (trophozoites mixed equally with cysts) were serially diluted from 1 × 106 to 0.3 × 105, placed on a 2-mm contact lens, and put into the mouse eye. After the eyelid was sutured, the occurrence of keratitis symptoms was observed from day 1 to 7 post-inoculation. In addition, to confirm the AK development, the genomic DNA was extracted from the infected mouse ocular tissue. Amplification of acanthamoebic 18S-rRNA gene by PCR using P-FLA primers was performed. In conclusion, the experimental mouse AK, characterized by typical hazy blurring and melting of mouse cornea, was developed from day 1 to 7 post-inoculation with 0.5 × 105 amoeba. PCR products from DNA samples of mouse eyeballs on day 1 to 7 were amplified to be same as 18S-rRNA gene of A. castellanii used a positive control. Subsequently, the amplified bands were identified as 18S-rDNA of A. castellanii, which showed 91–97 % homology, by BLAST search. To confirm the effect of the commercial lens solution, 0.5 × 105 amoebas (trophozoites mixed equally with cysts) were treated with solutions A or B for 1, 6, 12 or 24 h, and inoculated into mouse eye (previously established AK mouse model). Thereafter, AK development was observed for 7 days post-inoculation. AK
55
occurrence by A. castellanii treated with lens solution A or B for 1 or 6 h was not grossly observed on day 1 and 2, but the infection progressed gradually with a typical circulatory edema and keratitis on day 3, 5 and 7. Upon pre-treatments for 12 or 24 h, the development of AK was not observed for 1, 2 or 3 days, but progressed to corneal infection at 5 and 7 days. Finally, commercial contact lens solutions did not show the protective effect against AK pathogenesis, although they delayed the occurrence of AK.
In conclusion, it is suggested that the present AK mouse model is an important in vivo model for future studies.
56
REFERENCES
1. Abjani F, Khan NA, Yousuf FA, Siddiqui R: Targeting cyst wall is an effective strategy in improving the efficacy of marketed contact lens disinfecting solutions against Acanthamoeba castellanii cysts. Cont Lens Anterior Eye 39: 239-243, 2016
2. Aksozek A, McClellan K, Howard K, Niederkorn JY, Alizadeh H: Resistance of Acanthamoeba castellanii cysts to physical, chemical, and radiological conditions. J Parasitol 88: 621-623, 2002
3. Alizadeh H, He Y, McCulley JP, Ma D, Stewart GL, Via M, Haehling E, Niederkorn JY: Successful immunization against Acanthamoeba keratitis in a pig model. Cornea 14: 180-186, 1995
4. Alves D, Goncalves GS, Moraes AS, Alves LM, Carmo Neto JRD, Hecht MM, Nitz N, Gurgel-Goncalves R, Bernardes G, Castro AM, Chalita MR, Vinaud MC:
The first Acanthamoeba keratitis case in the Midwest region of Brazil: diagnosis, genotyping of the parasite and disease outcome. Rev Soc Bras Med Trop 51:
716-719, 2018
5. Auran JD, Starr MB, Jakobiec FA: Acanthamoeba keratitis. A review of the literature. Cornea 6: 2-26, 1987
6. Awwad ST, Petroll WM, McCulley JP, Cavanagh HD: Updates in Acanthamoeba keratitis. Eye Contact Lens 33: 1-8, 2007
57
7. Bacon AS, Frazer DG, Dart JK, Matheson M, Ficker LA, Wright P: A review of 72 consecutive cases of Acanthamoeba keratitis, 1984-1992. Eye (Lond) 7 ( Pt 6): 719-725, 1993
8. Behera HS, Satpathy G: Characterisation and expression analysis of trophozoite and cyst proteins of Acanthamoeba spp. isolated from Acanthamoeba keratitis (AK) patient. Mol Biochem Parasitol 205: 29-34, 2016
9. Booton GC, Visvesvara GS, Byers TJ, Kelly DJ, Fuerst PA: Identification and distribution of Acanthamoeba species genotypes associated with nonkeratitis infections. J Clin Microbiol 43: 1689-1693, 2005
10. Bouheraoua N, Gaujoux T, Goldschmidt P, Chaumeil C, Laroche L, Borderie VM: Prognostic factors associated with the need for surgical treatments in acanthamoeba keratitis. Cornea 32: 130-136, 2013
11. Brown TJ, Cursons RT, Keys EA: Amoebae from antarctic soil and water. Appl Environ Microbiol 44: 491-493, 1982
12. Byers TJ, Hugo ER, Stewart VJ: Genes of Acanthamoeba: DNA, RNA and protein sequences (a review). J Protozool 37: 17S-25S, 1990
13. Cao Z, Jefferson DM, Panjwani N: Role of carbohydrate-mediated adherence in cytopathogenic mechanisms of Acanthamoeba. J Biol Chem 273: 15838-15845, 1998
14. Centers for Disease C: Acanthamoeba keratitis associated with contact lenses--United States. MMWR Morb Mortal Wkly Rep 35: 405-408, 1986
58
15. Cerva L, Serbus C, Skocil V: Isolation of limax amoebae from the nasal mucosa of man. Folia Parasitol (Praha) 20: 97-103, 1973
16. Cho JH, Na BK, Kim TS, Song CY: Purification and characterization of an extracellular serine proteinase from Acanthamoeba castellanii. IUBMB Life 50:
209-214, 2000
17. Chung DI, Yu HS, Hwang MY, Kim TH, Kim TO, Yun HC, Kong HH: Subgenus classification of Acanthamoeba by riboprinting. Korean J Parasitol 36: 69-80, 1998
18. Cope JR, Collier SA, Schein OD, Brown AC, Verani JR, Gallen R, Beach MJ, Yoder JS: Acanthamoeba Keratitis among Rigid Gas Permeable Contact Lens Wearers in the United States, 2005 through 2011. Ophthalmology 123: 1435-1441, 2016
19. Corsaro D, Venditti D: Phylogenetic evidence for a new genotype of Acanthamoeba (Amoebozoa, Acanthamoebida). Parasitol Res 107: 233-238, 2010
20. Costas M, Griffiths AJ: Enzyme composition and the taxonomy of Acanthamoeba. J Protozool 32: 604-607, 1985
21. Daggett PM, Lipscomb D, Sawyer TK, Nerad TA: A molecular approach to the phylogeny of Acanthamoeba. Biosystems 18: 399-405, 1985
22. Dart JK, Saw VP, Kilvington S: Acanthamoeba keratitis: diagnosis and treatment update 2009. Am J Ophthalmol 148: 487-499 e482, 2009
59
23. De Jonckheere JF: Ecology of Acanthamoeba. Rev Infect Dis 13 Suppl 5: S385-387, 1991
24. Fears AC, Metzinger RC, Killeen SZ, Reimers RS, Roy CJ: Comparative in vitro effectiveness of a novel contact lens multipurpose solution on Acanthamoeba castellanii. J Ophthalmic Inflamm Infect 8: 19, 2018
25. Goldschmidt P, Degorge S, Benallaoua D, Saint-Jean C, Batellier L, Alouch C, Laroche L, Chaumeil C: New tool for the simultaneous detection of 10 different genotypes of Acanthamoeba available from the American Type Culture
25. Goldschmidt P, Degorge S, Benallaoua D, Saint-Jean C, Batellier L, Alouch C, Laroche L, Chaumeil C: New tool for the simultaneous detection of 10 different genotypes of Acanthamoeba available from the American Type Culture